Preimplantation genetic testing for a chr14q32 microdeletion in a family with Kagami-Ogata syndrome and Temple syndrome.

INTRODUCTION: Kagami-Ogata syndrome (KOS14) and Temple syndrome (TS14) are two disorders associated with reciprocal alterations within the chr14q32 imprinted domain. Here, we present a work-up strategy for preimplantation genetic testing (PGT) to avoid the transmission of a causative micro-deletion. METHODS: We analysed DNA from the KOS14 index case and parents using methylation-sensitive ligation-mediated probe amplification and methylation pyrosequencing. The extent of the deletion was mapped using SNP arrays. PGT was performed in trophectoderm samples in order to identify unaffected embryos. Samples were amplified using multiple displacement amplification, followed by genome-wide SNP genotyping to determine the at-risk haplotype and next-generation sequencing to determine aneuploidies. RESULTS: A fully methylated pattern at the normally paternally methylated IG-DMR and MEG3 DMR in the KOS14 proband, accompanied by an unmethylated profile in the TS14 mother was consistent with maternal and paternal transmission of a deletion, respectively. Further analysis revealed a 108 kb deletion in both cases. The inheritance of the deletion on different parental alleles was consistent with the opposing phenotypes. In vitro fertilisation with intracytoplasmatic sperm injection and PGT were used to screen for deletion status and to transfer an unaffected embryo in this couple. A single euploid-unaffected embryo was identified resulting in a healthy baby born. DISCUSSION: We identify a microdeletion responsible for multigeneration KOS14 and TS14 within a single family where carriers have a 50% risk of transmitting the deletion to their offspring. We show that PGT can successfully be offered to couples with IDs caused by genetic anomalies.

Genomic and phenotypic characterization of 404 individuals with neurodevelopmental disorders caused by CTNNB1 variants.

PURPOSE: Germline loss-of-function variants in CTNNB1 cause neurodevelopmental disorder with spastic diplegia and visual defects (NEDSDV; OMIM 615075) and are the most frequent, recurrent monogenic cause of cerebral palsy (CP). We investigated the range of clinical phenotypes owing to disruptions of CTNNB1 to determine the association between NEDSDV and CP. METHODS: Genetic information from 404 individuals with collectively 392 pathogenic CTNNB1 variants were ascertained for the study. From these, detailed phenotypes for 52 previously unpublished individuals were collected and combined with 68 previously published individuals with comparable clinical information. The functional effects of selected CTNNB1 missense variants were assessed using TOPFlash assay. RESULTS: The phenotypes associated with pathogenic CTNNB1 variants were similar. A diagnosis of CP was not significantly associated with any set of traits that defined a specific phenotypic subgroup, indicating that CP is not additional to NEDSDV. Two CTNNB1 missense variants were dominant negative regulators of WNT signaling, highlighting the utility of the TOPFlash assay to functionally assess variants. CONCLUSION: NEDSDV is a clinically homogeneous disorder irrespective of initial clinical diagnoses, including CP, or entry points for genetic testing.

Impaired proteostasis in rare neurological diseases.

Rare diseases are classified as such when their prevalence is 1:2000 or lower, but even if each of them is so infrequent, altogether more than 300 million people in the world suffer one of the ∼7000 diseases considered as rare. Over 1200 of these disorders are known to affect the brain or other parts of our nervous system, and their symptoms can affect cognition, motor function and/or social interaction of the patients; we refer collectively to them as rare neurological disorders or RNDs. We have focused this review on RNDs known to have compromised protein homeostasis pathways. Proteostasis can be regulated and/or altered by a chain of cellular mechanisms, from protein synthesis and folding, to aggregation and degradation. Overall, we provide a list comprised of above 215 genes responsible for causing more than 170 distinct RNDs, deepening on some representative diseases, including as well a clinical view of how those diseases are diagnosed and dealt with. Additionally, we review existing methodologies for diagnosis and treatment, discussing the potential of specific deubiquitinating enzyme inhibition as a future therapeutic avenue for RNDs.

Sporadic Creutzfeldt-Jakob disease with extremely long 14-year survival period.

BACKGROUND AND PURPOSE: Sporadic Creutzfeldt-Jakob disease is a rapidly progressing and highly variable neurodegenerative disease with heterogeneous clinical presentation and a median survival time from diagnosis to death of 4-6 months. METHODS: We report a rare case of a 61-year-old woman with a history of initially rapidly progressive dementia, with subsequent development of pyramidal and extrapyramidal signs and with an unusually long survival period of 14 years. Initial magnetic resonance imaging evaluation, single-photon emission computed tomography, and electroencephalogram did not show relevant alterations. RESULTS: The postmortem examination of the brain showed diffuse spongiform change, gliosis, and neuronal loss along with abnormal immunostaining of prion protein in the grey matter, especially in the cerebellum. Indirect PRNP genetic analysis was negative. CONCLUSIONS: This case is, to our knowledge, the sporadic Creutzfeldt-Jakob disease patient with the longest survival period ever documented. This surprisingly long duration highlights the importance of histopathological confirmation with brain autopsies for suspected cases, as the disease can easily be misdiagnosed in such slowly progressing cases.

What to consider when pseudohypoparathyroidism is ruled out: iPPSD and differential diagnosis.

BACKGROUND: Pseudohypoparathyroidism (PHP) is a rare disease whose phenotypic features are rather difficult to identify in some cases. Thus, although these patients may present with the Albright's hereditary osteodystrophy (AHO) phenotype, which is characterized by small stature, obesity with a rounded face, subcutaneous ossifications, mental retardation and brachydactyly, its manifestations are somewhat variable. Indeed, some of them present with a complete phenotype, whereas others show only subtle manifestations. In addition, the features of the AHO phenotype are not specific to it and a similar phenotype is also commonly observed in other syndromes. Brachydactyly type E (BDE) is the most specific and objective feature of the AHO phenotype, and several genes have been associated with syndromic BDE in the past few years. Moreover, these syndromes have a skeletal and endocrinological phenotype that overlaps with AHO/PHP. In light of the above, we have developed an algorithm to aid in genetic testing of patients with clinical features of AHO but with no causative molecular defect at the GNAS locus. Starting with the feature of brachydactyly, this algorithm allows the differential diagnosis to be broadened and, with the addition of other clinical features, can guide genetic testing. METHODS: We reviewed our series of patients (n = 23) with a clinical diagnosis of AHO and with brachydactyly type E or similar pattern, who were negative for GNAS anomalies, and classify them according to the diagnosis algorithm to finally propose and analyse the most probable gene(s) in each case. RESULTS: A review of the clinical data for our series of patients, and subsequent analysis of the candidate gene(s), allowed detection of the underlying molecular defect in 12 out of 23 patients: five patients harboured a mutation in PRKAR1A, one in PDE4D, four in TRPS1 and two in PTHLH. CONCLUSIONS: This study confirmed that the screening of other genes implicated in syndromes with BDE and AHO or a similar phenotype is very helpful for establishing a correct genetic diagnosis for those patients who have been misdiagnosed with "AHO-like phenotype" with an unknown genetic cause, and also for better describing the characteristic and differential features of these less common syndromes.

Familial Progressive Hyperpigmentation, Cutaneous Mastocytosis, and Gastrointestinal Stromal Tumor as Clinical Manifestations of Mutations in the c-KIT Receptor Gene.

BACKGROUND: Familial progressive hyperpigmentation (FPH) is an autosomal dominant disorder characterized by the appearance of hyperpigmented patches on the skin from early infancy that increase in size and number with age. METHODS: We report the clinical and molecular studies of an 11-year-old boy who had areas of hyperpigmentation since birth that had spread across his body as irregular hyperpigmented macules and papules, and include relevant history in family members. RESULTS: Affected members of his family shared a mutation in the c-KIT gene. All had progressive hyperpigmentation, in some cases accompanied by gastrointestinal stromal tumors and mastocytoma. There have been few reports of familial progressive hyperpigmentation together with systemic manifestations. CONCLUSIONS: Molecular analysis of c-KIT should be considered in the presence of FPH with systemic involvement.

Clinical and molecular analyses of Beckwith-Wiedemann syndrome: Comparison between spontaneous conception and assisted reproduction techniques.

Beckwith-Wiedemann syndrome (BWS) is an overgrowth syndrome characterized by an excessive prenatal and postnatal growth, macrosomia, macroglossia, and hemihyperplasia. The molecular basis of this syndrome is complex and heterogeneous, involving genes located at 11p15.5. BWS is correlated with assisted reproductive techniques. BWS in individuals born following assisted reproductive techniques has been found to occur four to nine times higher compared to children with to BWS born after spontaneous conception. Here, we report a series of 187 patients with to BWS born either after assisted reproductive techniques or conceived naturally. Eighty-eight percent of BWS patients born via assisted reproductive techniques had hypomethylation of KCNQ1OT1:TSS-DMR in comparison with 49% for patients with BWS conceived naturally. None of the patients with BWS born via assisted reproductive techniques had hypermethylation of H19/IGF2:IG-DMR, neither CDKN1 C mutations nor patUPD11. We did not find differences in the frequency of multi-locus imprinting disturbances between groups. Patients with BWS born via assisted reproductive techniques had an increased frequency of advanced bone age, congenital heart disease, and decreased frequency of earlobe anomalies but these differences may be explained by the different molecular background compared to those with BWS and spontaneous fertilization. We conclude there is a correlation of the molecular etiology of BWS with the type of conception. © 2016 Wiley Periodicals, Inc.

Choosing the Best Tissue and Technique to Detect Mosaicism in Fibrous Dysplasia/McCune-Albright Syndrome (FD/MAS).

GNAS-activating somatic mutations give rise to Fibrous Dysplasia/McCune-Albright syndrome (FD/MAS). The low specificity of extra-skeletal signs of MAS and the mosaic status of the mutations generate some difficulties for a proper diagnosis. We studied the clinical and molecular statuses of 40 patients referred with a clinical suspicion of FD/MAS to provide some clues. GNAS was sequenced using both Sanger and Next-Generation Sequencing (NGS). We were able to identify the pathogenic variants in 25% of the patients. Most of them were identified in the affected tissue, but not in blood. Additionally, NGS demonstrated the ability to detect more patients with mosaicism (8/34) than Sanger sequencing (4/39). Even if in some cases, the clinical information was not complete, we confirmed that, as in previous works, when the patients were young children with a single manifestation, such as hyperpigmented skin macules or precocious puberty, the molecular diagnosis was usually negative. In conclusion, as FD/MAS is caused by mosaic variants, it is essential to use sensitive techniques that allow for the detection of low percentages and to choose the right tissue to study. When not possible, and due to the low positive genetic rate, patients with FD/MAS should only be genetically tested when the clinical diagnosis is really uncertain.

Colonic adenomatous polyp with florid presence of monoclonal lambda Russell bodies: Case report and etiopathogenic hypothesis.

Russell bodies (RBs) are round eosinophilic intracytoplasmic inclusions formed by condensed immunoglobulins in mature plasma cells, which are called Mott cells. These cells are rarely found in the gastric tract, with even less cases reported in the colorectal region. There are still many questions about this event, as it is still unknown the relationship between the agents reported of increasing the probability of appearance of these cells and the generation of RBs. In this case report we describe the fifth patient presenting an infiltration of Mott cells in a colorectal polyp, being the second case with a monoclonal origin without a neoplastic cause, and the first one monoclonal for lambda. A comparison with previously similar reported cases is also done, and a possible etiopathogenic hypothesis proposed.

A Protein Misfolding Shaking Amplification-based method for the spontaneous generation of hundreds of bona fide prions.

Prion diseases are a group of rapidly progressing neurodegenerative disorders caused by the misfolding of the endogenous prion protein (PrPC) into a pathogenic form (PrPSc). This process, despite being the central event underlying these disorders, remains largely unknown at a molecular level, precluding the prediction of new potential outbreaks or interspecies transmission incidents. In this work, we present a method to generate bona fide recombinant prions de novo, allowing a comprehensive analysis of protein misfolding across a wide range of prion proteins from mammalian species. We study more than 380 different prion proteins from mammals and classify them according to their spontaneous misfolding propensity and their conformational variability. This study aims to address fundamental questions in the prion research field such as defining infectivity determinants, interspecies transmission barriers or the structural influence of specific amino acids and provide invaluable information for future diagnosis and therapy applications.

Genome-wide allelic methylation analysis reveals disease-specific susceptibility to multiple methylation defects in imprinting syndromes.

Genomic imprinting is the parent-of-origin-specific allelic transcriptional silencing observed in mammals, which is governed by DNA methylation established in the gametes and maintained throughout the development. The frequency and extent of epimutations associated with the nine reported imprinting syndromes varies because it is evident that aberrant preimplantation maintenance of imprinted differentially methylated regions (DMRs) may affect multiple loci. Using a custom Illumina GoldenGate array targeting 27 imprinted DMRs, we profiled allelic methylation in 65 imprinting defect patients. We identify multilocus hypomethylation in numerous Beckwith-Wiedemann syndrome, transient neonatal diabetes mellitus (TNDM), and pseudohypoparathyroidism 1B patients, and an individual with Silver-Russell syndrome. Our data reveal a broad range of epimutations exist in certain imprinting syndromes, with the exception of Prader-Willi syndrome and Angelman syndrome patients that are associated with solitary SNRPN-DMR defects. A mutation analysis identified a 1 bp deletion in the ZFP57 gene in a TNDM patient with methylation defects at multiple maternal DMRs. In addition, we observe missense variants in ZFP57, NLRP2, and NLRP7 that are not consistent with maternal effect and aberrant establishment or methylation maintenance, and are likely benign. This work illustrates that further extensive molecular characterization of these rare patients is required to fully understand the mechanism underlying the etiology of imprint establishment and maintenance.

Simultaneous hyper- and hypomethylation at imprinted loci in a subset of patients with GNAS epimutations underlies a complex and different mechanism of multilocus methylation defect in pseudohypoparathyroidism type 1b.

Most patients with pseudohypoparathyroidism type 1b (PHP-1b) display a loss of imprinting (LOI) encompassing the GNAS locus resulting in PTH resistance. In other imprinting disorders, such as Russell-Silver or Beckwith-Wiedemann syndrome, we and others have shown that the LOI is not restricted to one imprinted locus but may affect other imprinted loci for some patients. Therefore, we hypothesized that patients with PHP-1b might present multilocus imprinting defects. We investigated, in 63 patients with PHP-1b, the methylation pattern of eight imprinted loci: GNAS, ZAC1, PEG1/MEST, ICR1, and ICR2 on chromosome 11p15, SNRPN, DLK1/GTL2 IG-DMR, and L3MBTL1. We found multilocus imprinting defects in four PHP-1b patients carrying broad LOI at the GNAS locus (1) simultaneous hypermethylation at L3MBTL1 differentially methylated region 3 (DMR3), and hypomethylation at PEG1/MEST DMR (n = 1), (2) hypermethylation at the L3MBTL1 (DMR3) (n = 1) and at the DLK1/GTL2 IG-DMR (n = 1), and (3) hypomethylation at the L3MBTL1 DMR3 (n = 1). We suggest that mechanisms underlying multilocus imprinting defects in PHP-1b differ from those of other imprinting disorders having only multilocus loss of methylation. Furthermore, our results favor the hypothesis of "epidominance", that is, the phenotype is controlled by the most severely affected imprinted locus.

Recessive mutations in the INS gene result in neonatal diabetes through reduced insulin biosynthesis.

Heterozygous coding mutations in the INS gene that encodes preproinsulin were recently shown to be an important cause of permanent neonatal diabetes. These dominantly acting mutations prevent normal folding of proinsulin, which leads to beta-cell death through endoplasmic reticulum stress and apoptosis. We now report 10 different recessive INS mutations in 15 probands with neonatal diabetes. Functional studies showed that recessive mutations resulted in diabetes because of decreased insulin biosynthesis through distinct mechanisms, including gene deletion, lack of the translation initiation signal, and altered mRNA stability because of the disruption of a polyadenylation signal. A subset of recessive mutations caused abnormal INS transcription, including the deletion of the C1 and E1 cis regulatory elements, or three different single base-pair substitutions in a CC dinucleotide sequence located between E1 and A1 elements. In keeping with an earlier and more severe beta-cell defect, patients with recessive INS mutations had a lower birth weight (-3.2 SD score vs. -2.0 SD score) and were diagnosed earlier (median 1 week vs. 10 weeks) compared to those with dominant INS mutations. Mutations in the insulin gene can therefore result in neonatal diabetes as a result of two contrasting pathogenic mechanisms. Moreover, the recessively inherited mutations provide a genetic demonstration of the essential role of multiple sequence elements that regulate the biosynthesis of insulin in man.

[The enigma of Henry IV's disease: Did he suffer from McCune-Albright syndrome/fibrous dysplasia?] / El enigma de la enfermedad de Enrique IV, rey de Castilla: ¿padeció síndrome de McCune-Albright/displasia fibrosa?

BACKGROUND: Henry IV King of Castile, last king of the Trastámara dynasty, was the brother of Isabella the Catholic. He is known as "the impotent". Based on previous descriptions by historians and biographers, Gregorio Marañón in 1922 described him as "eunuchoid dysplastic with acromegalic reaction and clear schizoid features". METHODS: In 1946, a post-mortem inspection was carried out on the mummified corpse found in the Monastery of Guadalupe. A written document and some photographs were recorded. We have collected the signs and symptoms described and applied the international classification of diseases recommended by the World Health Organisation, ICD11-2023. We have relied on the coins issued in the money of Henry IV, on which we have identified enlargement of the thyroid gland. RESULTS: With the data available at this time, we suggest that Henry IV most probably suffered from: facial and polyostotic bone dysplasia, kyphosis, limb limping, multiple endocrine disorders, acromegaly with macrognatia, nodular thyroid disease, malodorous diaphoresis, erectile dysfunction, hypospadias, abnormal sexual development, "feminoid pelvis", abdominal colic, oligodontia and dental displacement. It is possible that he also suffered from: precocious puberty, renal lithiasis with debilitating phosphaturia, carpal tunnel, thrombopenia and growth hormone-producing pituitary hyperplasia or adenoma. CONCLUSION: We suggest that Henry IV may have suffered from McCune-Albrigth syndrome associated with fibrous dysplasia, a rare disease due to gain-of-function mutations in the GNAS gene.

New pathogenic variant in DLX5: New clues for a clinical spectrum from split-hand-foot malformation to fibular aplasia, tibial campomelia and oligosyndactyly.

Introduction: FATCO (Fibular Aplasia, Tibial Campomelia and Oligosyndactyly) is a very infrequent skeletal dysplasia classified within the limb hypoplasia-reduction defects group whose genetic cause has not yet been identified. The advent of next-generation sequencing is enabling the diagnosis of diseases with no previously known genetic cause. Methods: We performed a thorough autopsy on a fetus whose pregnancy was legally terminated due to severe malformations detected by ultrasound. A trio exome was run to identify the genetic cause and risk of recurrence. Previous literature of similar cases was systematically searched. Results: Anatomopathological analyses revealed complete fibular aplasia, shortened and campomelic tibia, absent ankle joint, club right foot and a split foot malformation, leading to the diagnosis of FATCO. Exome sequencing showed that the female fetus carried a de novo nonsense variant in DLX5. The literature search permitted the collection of information on 43 patients with FATCO, the majority of whom were males diagnosed postnatally. In most cases, lower limbs were affected exclusively, but in 39.5% of cases the upper limbs were also affected. Conclusion: The pathologies associated with DLX5 variants encompass a wide spectrum of manifestations ranging from abnormalities exclusively in the hands and feet to long bones such as the tibia and fibula.

Incidental finding at methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA): how to proceed?

Introduction: Since the advent of new generation sequencing, professionals are aware of the possibility of obtaining findings unrelated to the pathology under study. However, this possibility is usually forgotten in the case of studies aimed at a single gene or region. We report a case of a 16-month-old girl with clinical suspicion of Silver-Russell syndrome (SRS). Methods: Following the international SRS consensus, methylation alterations and copy number variations (CNVs) at 11p15 region and maternal uniparental disomy of chromosome 7 were analysed and discarded by MS-MLPA. Results: Unexpectedly, the 11p15 region MS-MLPA showed a decrease in the signal of a copy number reference probe. Deletions affecting a single probe are inconclusive. So, we faced the ethical dilemma of whether it was appropriate to confirm this alteration with independent techniques and to offer a diagnostic possibility that was in no way related to clinical suspicion. Fortunately, in this particular case, the informed consent had not been specific to a particular pathology but to any disorder associated with growth failure. Performed alternative studies allowed the final diagnosis of 22q deletion syndrome. Conclusion: We demonstrate the importance of informing patients about the possibility of obtaining incidental findings in genetic techniques (not only in next generation sequencing) during pre-test genetic counselling consultations. In addition, we highlight the relevance of including in the informed consent the option of knowing these unexpected incidental findings as in some cases, this will help to elucidate the definitive diagnosis and provide the correct follow-up and treatment.

C9ORF72 Gene GGGGCC Hexanucleotide Expansion: A High Clinical Variability from Amyotrophic Lateral Sclerosis to Frontotemporal Dementia.

The expanded GGGGCC hexanucleotide repeat (HRE) in the non-coding region of the C9ORF72 gene (C9ORF72-HRE) is the most common genetic cause of familial forms of amyotrophic lateral sclerosis (ALS), FTD, and concurrent ALS and FTD (ALS-FTD), in addition to contributing to the sporadic forms of these diseases. Both syndromes overlap not only genetically, but also sharing similar clinical and neuropathological findings, being considered as a spectrum. In this paper we describe the clinical-genetic findings in a Basque family with different manifestations within the spectrum, our difficulties in reaching the diagnosis, and a narrative review, carried out as a consequence, of the main features associated with C9ORF72-HRE. Family members underwent a detailed clinical assessment, neurological examination, and genetic analysis by repeat-primed PCR. We studied 10 relatives of a symptomatic carrier of the C9ORF72-HRE expansion. Two of them presented the expansion in the pathological range, one of them was symptomatic whereas the other one remained asymptomatic at 72 years. Given the great intrafamilial clinical variability of C9ORF72-HRE, the characterization of patients and family members with particular clinical and genetic subgroups within ALS and FTD becomes a bottleneck for medication development, in particular for genetically focused medicines for ALS and FTD.